Inadequate sensitivity of existing XBP1 stress sensing systems can be overcome by improving the efficiency of unconventional splicing of xbp1 mRNA. Recent reports regarding the cellular localization of XBP1/HAC1 mRNA during its splicing allowed construction of a highly sensitive HG stress indicator that can visualize the activation of IRE1/XBP1 pathway at the third instar larval stage during normal Drosophila development. Several types of cells in the organs where IRE1/XBP1 activity was detected are known for having high secretory capacity (Sone, 2013).

In the larval brain, significant IRE1/XBP1 activity was found in glial cells. While glia had been originally thought to function as the structural support cells in the nervous system, it has been revealed that they play several important roles in the development and homeostasis of the nervous system. In Drosophila, glial cells are classified into three classes (surface-, cortex-, and neuropil-associated glia), each of which is subdivided further morphologically. Whether IRE1/XBP1 active glia is restricted to only a subtype of those glia, or more broadly, is currently under investigation (Sone, 2013).

In mammals, oligodendrocytes in the central nerve system and Schwann cells in the peripheral nerve system myelinate axons by producing a large amount of myelin membrane proteins, cholesterol, and membrane lipids through the secretory pathway. Recent reports suggested that ER stress in myelinating cells is important in the pathogenesis of various disorders of myelin. Neuropil glia and peripheral glia in Drosophila are the counterparts of oligodendrocytes and Schwann cells, respectively. Therefore, these cells are the candidates that show constitutive IRE1/XBP1 activity. Although Drosophila glia do not generate myelin sheaths, they form multi-layered membrane sheaths around neurons that are morphologically similar to the myelin sheaths in mammals. Thus, it is possible that the IRE1/XBP1 active glia protect neurons from their deterioration through this ensheathment, thereby contributing to brain homeostasis. Further studies are expected to be informative as to the pathological significance of IRE1/XBP1 functions in human glia (Sone, 2013).

IRE1/XBP1 pathway does not appear to be active in neuron. However, the possibility of neuronal IRE1/XBP1 activation in the brain was not excluded. In fact, slight neuronal IRE1/XBP1 activity was occasionally observed in the ventral nerve cord during repeated experiments. In this study, it is concluded that in the third instar larval brain, the IRE1/XBP1 pathway is predominantly activated in glia while the activation is not detectable in neurons (Sone, 2013).

The importance of IRE1/XBP1 activity in the gut has already been studied in Caenorhabditis elegans and mammals. Intra-tissue distribution of IRE1/XBP1 activity was detected in the proventriculus region of the gut. In the larval midgut and hindgut, an irregular distribution of IRE1/XBP1 active cells was observed. These were not entero-endocrine cells, as they did not colocalize with anti-Prospero antibody that marks those cells. Secretory intestinal cells in the midgut other than entero-endocrine cells including the intestinal stem cells are possible candidates for these IRE1/XBP1 active cells (Sone, 2013).

IRE1/XBP1 activity in the fly Malpighian tubules (analogous to the kidney in mammals) was also unexpected. The activity was detected throughout the organ, but not all of the cells were IRE1/XBP1 active. Although the Malpighian tubules are attached at the junction of the midgut and the hindgut, they are morphologically and functionally independent from both of them. Identification of the IRE1/XBP1 active cells in the gut and the Malpighian tubules might reflect a shared physiological function of both organs. One possible shared function may be the selective uptake of the essential molecules, including several metal ions, from the contents passing through those organs. IRE1/XBP1 pathway might regulate the function of some transporter channels in these organs. Drosophila Malpighian tubules are expected to be one of the models for the mammalian diabetic kidney diseases that are associated with UPR activation (Sone, 2013).

In this study, IRE1/XBP1 activity was also detected in the trachea. Previous reports suggest its relevance to glial IRE1/XBP1 activity. One of them showed that tracheal development in Drosophila brain was controlled by signals from glia. According to the report, the branches of cerebral trachea grow around the neuropile. If IRE1/XBP1 active glia were neuropile-associated glia, assessing IRE1/XBP1 activity at neuropile-associated glia is likely reveal the shared physiological function of IRE1/XBP1 pathway between brain and trachea. The other report, using embryonic trachea, indicated that the proper combination of secretory activity and endocytotic activity was importaAnt for the maturation of trachea as an airway. In tracheal maturation, Sar1, one of the core COPII proteins, was required for the secretion of protein, the luminal matrix assembly, and the following expansion of tube diameter to avoid the clogging of protein, while Rab5, the small GTPase that regulates the early stage of endocytosis, was required for the clearance of deposited materials in the lumen. It can be predicted that, even in larval trachea, IRE1/XBP1 pathway plays a crucial role in tracheal maturation by supplying the properly folded proteins to the transport machinery. In that case, in view of second instar larval lethality of xbp1-/- hypomorph mutant, it could also be hypothesized that the tracheal maturation/maintenance is still important for larval lethality, in addition to its importance for the embryonic development (Sone, 2013).

IRE1/XBP1 activity in the salivary gland has already been reported in a previous study (Souid, 2007). The salivary gland is commonly used for the determination of the subcellular localization of the protein in Drosophila cells due to its morphological features. The nuclear localization of HG indicator, XBP1-EGFP molecule, was clearly indicated. In addition, weak IRE1/XBP1 activity was detected in the fat body; it was attached to the salivary gland. Generally, the Drosophila fat body, which is equivalent to mammalian adipose tissue, functions as the organ for energy/lipid storage and is distributed throughout the larval body (Sone, 2013).

In addition to the larval tissues, IRE1/XBP1 activity was analyzed in the adult male reproductive organs. Though the previous RT-PCR study by Souid (2007) suggested the activity in the testis, the areas where IRE1/XBP1 activity was detected were the accessory glands and a limited area of the testis close to the testicular duct. In the accessory gland, seminal fluid containing several hormones, which facilitate reproductive traits such as sperm transfer, sperm storage, female receptivity, ovulation, and oogenesis, are produced and secreted. There are two morphologically distinct secretory cell types in Drosophila accessory gland. Ninety-six percent of the secretory cells are categorized as main cells and the others are secondary cells. Based on the intra-tissue distribution of IRE1/XBP1 active cells in the accessory gland, the active cells are likely to be main cells. Since each of these cell types expresses a unique set of genes, the confirmation of IRE1/XBP1 active cell type is expected to allow ingnarrow down the proteins related to IRE1/XBP1 activity. IRE1/XBP1 pathway is likely to function, to some extent, in maintaining proper fertility (Sone, 2013).

On the other hand, a possibility is considered that the EGFP signal detected in each organ might not necessarily reflect the unconventional splicing of xbp1-EGFP mRNA. Higher concentrations of the spliced xbp1-EGFP mRNA and resulting XBP1-EGFP in the cells induced by the Gal4/UAS system might cause the artifactual EGFP signal. The possibility was excluded that the EGFP signal in this study was detected independently of the unconventional splicing, based on the results in this study and the following reasoning (Sone, 2013).

There are two possible molecular mechanisms that cause the artifactual EGFP signal which is not derived from the unconventional splicing of xbp1-EGFP mRNA. One is the generation of EGFP or abnormal EGFP fusion proteins, resulting from translation initiation at the start codon of the EGFP coding sequence or at ATG codons coding Met residues in XBP1(s), respectively. The other is the proteolytic digestion of XBP1-EGFP fusion protein at the junction of XBP1 and EGFP portions. Both of these are prone to happen upon overexpression of fusion proteins in cells. In particular, the proteolytic digestion is often observed in the overexpression of GST fusion protein in Escherichia coli (Sone, 2013).

In this system, there is no nuclear localization signal (NLS) on the EGFP molecule. In contrast, xbp1 gene carries a NLS coding sequence located upstream of the unconventional splice site. There are a total of 11 ATG codons that code the Met residues of XBP1(s) molecule. Eight of the ATG codons are located downstream of NLS coding sequence. Therefore, due to the lack of NLS, both EGFP and the EGFP fusion proteins using these eight ATG codons as start codons should diffuse all over the cell upon their synthesis, if they were generated. EGFP signal was detected exclusively in the nucleus in the salivary gland, which is often used for the analysis of the cellular localization of the proteins in Drosophila. Hence, it is not reasonable to conclude that either EGFP or the possible eight EGFP fusion proteins above were expressed in the cells. Only the EGFP fusion proteins that use the other three ATG codons that are upstream of the NLS as start codons should be synthesized upon unconventional splicing and localized in the nucleus. The estimated molecular weights of those fusion proteins are 73.7, 74.3, and 80.0 kDa, respectively. Only a significant single band that represented the intact XBP1-EGFP (80.0 kDa) was detected in the S2 cell extract, in which XBP1-EGFP was overexpressed through the Gal4/UAS system. Therefore, it is concluded that the EGFP fusion protein synthesized in this study was the intact XBP1-EGFP (Sone, 2013).

Additionally, there are several ATG codons that are located upstream of the unconventional splice site and are in frame with EGFP coding sequence on unspliced xbp1 mRNA. However, inside the 23 bp of unconventional-spliced fragment, there is a TGA stop codon that is also in frame with EGFP coding sequence. Even if the translation initiated from these start codons, the synthesis of these products should be terminated at this TGA stop codon before the ribosome would reach the EGFP coding region (Sone, 2013).

Regarding the proteolytic digestion of XBP1-EGFP fusion protein, the resulting EGFP should diffuse all over the cell upon its synthesis due to the lack of NLS. Therefore, the possibility of the proteolytic digestion is also excluded based on the same reasoning as above. Taken together, it is concluded that the detected EGFP signal in this study exclusively reflected the occurrence of unconventional splicing of xbp1-EGFP mRNA (Sone, 2013).

In summary, this study improved the sensitivity of the XBP1 stress sensing system and newly identified several organs where IRE1/XBP1 pathway is constitutively activated under normal physiological conditions. In particular, in the larval brain, significant glial specific activation was detected. The improved system is expected to provide with a number of clues to reveal the molecular mechanisms underlying the normal development and homeostasis controlled by IRE1/XBP1 pathway (Sone, 2013).

Exploring the Conserved role of MANF in the unfolded protein response in Drosophila melanogaster

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. This study discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach genetic interactions was found between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. These data suggest a role for Manf in the regulation of Drosophila UPR (Lindstrom, 2016).

The accumulation of unfolded or misfolded proteins causes disturbances in endoplasmic reticulum (ER) homeostasis, a phenomenon referred to as ER stress. ER stress in turn activates the unfolded protein response (UPR). In order to overcome ER stress, UPR leads to attenuation of protein synthesis, enhancement of degradation of unfolded proteins, and activation of specific signalling cascades. These events aim to reduce the overall protein load in the ER and to enhance the protein folding capacity by selective transcription of chaperones. UPR is activated through three signalling cascades by ER transmembrane sensor proteins PERK (PRKR-like endoplasmic reticulum kinase), IRE1 (inositol requiring enzyme 1), and ATF6 (activating transcription factor 6). All of these three proteins are maintained inactive in normal cellular status by binding to the major ER chaperone GRP78/BiP (Glucose-regulated protein 78/Binding immunoglobulin protein). Upon ER stress, GRP78 is dissociated from the sensor proteins which are subsequently activated. The most ancient of these signalling cascades is mediated by IRE1, the sole branch of UPR identified in Saccharomyces cerevisiae. IRE1 has kinase activity and endoribonuclease activity needed for degradation of mRNAs in order to relieve the protein synthesis load. IRE1 is also responsible for the unconventional splicing and thus activation of transcription factor XBP1 (X-box Binding Protein-1), a positive regulator of ER chaperone and other UPR related gene expression. Activated PERK attenuates overall protein synthesis through phosphorylating and thus inhibiting EIF2α (eukaryotic translation initiation factor 2, subunit 1 α). However, the decreased activation of EIF2α results in an upregulated translation of specific target mRNAs including ATF4 (activating transcription factor 4). The third signalling pathway is mediated through ATF6, a transcription factor activated by its cleavage and translocation to the nucleus (Lindstrom, 2016).

In Drosophila, both IRE1- and PERK-mediated UPR signalling cascades are conserved. The amino acid sequence of the Drosophila homologue of ATF6 is highly similar to its mammalian counterpart, but experimental evidence for its involvement in Drosophila UPR is lacking. Similar to mammals, the expression of Drosophila homologue of GRP78, Hsc3 (Heat shock protein cognate 3), is upregulated upon induced ER stress in Xbp1-dependent manner but no biochemical data are available to show its association with ER stress sensor proteins (Lindstrom, 2016).

The MANF/CDNF family of neurotrophic factors was first characterized based on its trophic function on dopaminergic neurons in vitro and in vivo. When injected into the brain, recombinant mammalian MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) and CDNF (Cerebral Dopamine Neurotrophic Factor) protect and repair dopaminergic neurons in toxin-induced rodent models of Parkinson's disease (PD) in vivo. The sole Drosophila homologue, DmManf, is expressed in and secreted from glial cells and supports the dopaminergic system in non-cell-autonomous manner (Palgi, 2009). The role of MANF as an extracellular trophic factor is further supported by the evidence that mammalian MANF is protective against ischemic injury in both neurons and cardiomyocytes (Airavaara, 2010; Glembotski, 2012). However, the biology of MANF is not thoroughly understood. Intriguingly, MANF localizes to the ER and has a protective role against ER stress in vitro and in vivo. Additionally, mammalian MANF binds GRP78 in Ca2+-dependent manner in vitro and this binding may regulate MANF secretion. MANF can be retained in the ER by its C-terminal signal sequence, RTDL in human and RSEL in Drosophila (Glembotski, 2012; Lindström, 2013). Experimental evidence suggests that mammalian MANF interacts with KDEL-R [KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor] and that the C-terminal RTDL sequence of MANF is responsible for this interaction. The relevance of KDEL-R as a mediator of the functions of MANF has not been explored in vivo, yet. Recently, MANF was also shown to regulate the expression of ER-resident protein CRELD2 (Lindstrom, 2016).

Both in vivo and in vitro studies have shown that MANF is upregulated after chemically induced ER stress and by misfolded mutant proteins accumulating in the ER. Mammalian MANF expression is activated upon ER stress by ATF6 and XBP1 through an ER stress response element II found in the promoter region of MANF. Based on a knockout mouse model, MANF was found to be essential for the survival of pancreatic β-cells and its loss resulted in severe diabetes due to reduction of beta cell mass and activation of UPR in the pancreatic islets. The protective role against 6-OHDA induced and ischemic neuronal damage has been suggested to rise from the ER-related functions of MANF as these processes have been shown to induce ER stress (Lindstrom, 2016).

In Drosophila, the loss of DmManf is associated with upregulated expression of genes involved in UPR (Palgi, 2012). Additionally, the overexpression of DmManf resulted in downregulation of several UPR-related genes (Palgi, 2012). This study shows that, similar to mammalian MANF, the expression of DmManf is induced in response to ER stress in vitro. Further, transgenic approaches for gene silencing in vivo were applied to reveal genetic interactions between DmManf and genes with known functions in the maintenance of ER homeostasis and in UPR (Lindstrom, 2016).

Increasing evidence indicates that ER stress and UPR play a major role in variety of human diseases including diabetes mellitus and neurodegenerative disorders. MANF is a secreted protein (Palgi, 2009; Lindholm, 2008), but also localizes to the ER and has a role in mammalian UPR (Mizobuchi, 2007; Palgi, 2012). This study examined the role of DmManf in UPR in the Drosophila model. Upregulation of MANF mRNA expression by ER stress-inducing agents was shown to be conserved in Drosophila S2 cells. Additionally, genetic interaction between DmManf and genes known to function in the ER and UPR were shown (see A simplified presentation of UPR and genetic interactions discovered for Drosophila Manf; Lindstrom, 2016).

One of the interacting partners was Hsc3, the Drosophila homologue of mammalian chaperone GRP78. The silencing of Hsc3 in the wing resulted in an abnormal wing phenotype in wild type background. This wing phenotype was stronger in DmManf-overexpressing background. In cultured mammalian cells MANF has been shown to bind GRP78 in Ca2+-dependent manner and the loss of interaction between mammalian MANF and GRP78 was associated with increased secretion of MANF (Glembotski, 2012). In line, the knockdown of Hsc3 could lead to increased secretion of DmManf and lead to deprivation of intracellular DmManf. In a previous study, it was noticed that the deletion of ER retention signal RSEL increased the secretion of DmManf in S2 cells and decreased its functionality in rescue experiments in vivo (Lindström, 2013). Based on the physical interaction found between mammalian MANF and GRP78, the simultaneous overexpression of DmManf and knockdown of Hsc3 could also result in the abundant DmManf binding the residual Hsc3 and preventing other important cellular functions of Hsc3. Alternatively, the loss of Hsc3 could lead to decreased protein folding capacity in the ER and activation of UPR. The vast amount of DmManf protein could exhaust this already disturbed cellular state (Lindstrom, 2016).

In previous studies, mammalian MANF has been suggested to have chaperone-like functions, e.g. by binding unfolded proteins in vitro but the putative chaperone activity remains unconfirmed. The major ER chaperone Hsc3 and DmManf clearly have distinct roles as either the overexpression or the loss of one could not complement for the loss of the other. However, the current study indicates that the interaction between MANF and GRP78 (Glembotski, 2012) is conserved. In future, the functional significance of this intriguing interaction deserves to be addressed in detail (Lindstrom, 2016).

DmManf genetically interacted with PEK/PERK, an ER stress sensor protein. Similar to the silencing of Hsc3, simultaneous overexpression of DmManf worsened the phenotypes observed in PEK knockdown flies. Previous studies have indicated functional conservation of PERK in Drosophila and mammals. The Drosophila homologue to ATF4, the downstream target of activated PERK and selectively upregulated by UPR, showed no genetic interaction with DmManf in this study. It was previously shown that the abolishment of both zygotic and maternal DmManf resulted in increased phosphorylation of eIF2α, another molecular marker used for detecting ER stress (Palgi, 2012). This study abolished only the zygotic DmManf while maternal DmManf was still present. The loss of zygotic DmManf alone did not induce UPR when evaluated by other readouts, i.e. increased Hsc3 mRNA level and splicing of Xbp1. Although the zygotic DmManfΔ96 mutant larvae show only low amount of persisting maternal
DmManf mRNA and protein, it could be sufficient to prevent the induction of UPRl (Lindstrom, 2016).

Additionally, a genetic interaction was discovered between DmManf and Xbp1, a transcription factor mainly responsible for the regulation of UPR-induced genes. Upon UPR, the mRNA of Xbp1 is spliced by IRE1 and translated into a transcriptional activator of chaperone expression in response to the increased protein folding demand. According to previous studies, the spliced form of Xbp1 could mediate the UPR-induced upregulation of MANF in mammals. MANF has been suggested to have protective role against ER stress. During normal development, ER stress is detected in the secretory cells and the silencing of Xbp1 disturbs this developmental ER stress. Both mammalian and Drosophila MANF has been shown to have especially high expression levels in secretory tissues. Overexpression of DmManf increased Xbp1 mRNA level but the knockdown of Xbp1 did not affect DmManf expression levels. Also, the mRNA levels of Hsc3 were not upregulated in Xbp1 knockdown larvae. This could indicate the lack of transcriptional activation of DmManf and Hsc3 expression by Xbp1s in Xbp1-knockdown larvae. Therefore, knockdown of Xbp1 could compromise the regulation of DmManf expression in the developmental ER stress and deteriorate its function in the secretory cells (Lindstrom, 2016).

ERAD is a cellular process aiming to clear out the unfolded and misfolded proteins from the ER. According to previous transcriptome analysis, sip3 was downregulated in DmManfΔ96 mutant larvae (Palgi, 2012). This study also found a genetic interaction between DmManf and sip3. Sip3 encodes a homologue to mammalian ER resident E3 ubiquitin ligase synoviolin/HRD1 with specific function in ERAD. Mammalian MANF is upregulated by ERSE-II (ER stress response element II) found in its promoter region. Interestingly, ERSE-II is also found in ERAD-related components HERPUD1 (homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1, also known as HERP) and VIMP (VCP-interacting membrane protein, also known as selenoprotein S). ERSE-II has been hypothesized to regulate the protein quality control and degradation of misfolded proteins during ER stress suggesting that MANF could also have a role in these functions (Lindstrom, 2016).

Surprisingly, overexpression of DmManf led to enhanced phenotypes in flies of which a UPR-related gene was knocked down. Thus far, overexpression of DmManf with any GAL4 driver tested has never resulted in a detectable phenotype or altered viability. According to a previous microarray analysis, DmManf overexpression led to downregulation of UPR-related genes (Palgi, 2012). This suggests that the overexpression of DmManf would disturb UPR signalling. Hypothetically, wild type background cells would be able to deal with the increased DmManf expression and the subsequent downregulation of UPR-related genes whereas the additional knockdown of an important component of UPR, e.g. Hsc3, PEK or Xbp1, could compromise the cell homeostasis (Lindstrom, 2016).

An alternative explanation for these observations in interaction studies between UPR genes and DmManf would be that DmManf is actually a substrate for UPR. Then, the abundant expression of DmManf by UAS/GAL4 would rather model the effects of increased overall protein synthesis in ER than indicate specific ER-related functions for DmManf. DmManf enters the secretory pathway (Palgi, 2009) and its ectopic expression may cause stress to the protein folding machinery in the ER. Although the Xbp1 mRNA level was increased, the expression of Hsc3 was not altered indicating that overexpression of DmManf induces mild UPR. However, no similar effects were seen with overexpression of membrane-directed GFP suggesting that the observed phenomena were specific for DmManf (Lindstrom, 2016).

The previous microarray study found that the total loss of DmManf is associated with upregulated expression of genes involved in UPR (Palgi, 2012). However, in the current study the mRNA levels of Hsc3 and Xbp1 were mildly decreased in DmManf mutant larvae. In the previous study, transcriptome analysis was done from the embryonic DmManf mutants lacking both maternal and zygotic DmManf. In the current study, RNA was collected from zygotic DmManf mutants with the persisting maternal DmManf mRNA and protein. The maternal DmManf is apparently sufficient to prevent induction of UPR and upregulation of UPR related genes (Lindstrom, 2016).

This work provides evidence for the contribution of DmManf in Drosophila UPR. Further biochemical studies on the interaction between DmManf and UPR genes in Drosophila are needed to elucidate the details of this process (Lindstrom, 2016).

Regulation of axon regeneration by the RNA repair and splicing pathway

Mechanisms governing a neuron's regenerative ability are important but not well understood. This study has identified Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. This study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma (Song 2015).

Failure of damaged axons to regenerate is the primary cause of permanent disabilities after CNS injury and the irreversible neurologic dysfunction of neurodegenerative diseases. The ability of a neuron to regenerate its axon after trauma is governed by the interaction between its intrinsic growth capacity and the local environment. Notwithstanding the discoveries of extracellular factors and intrinsic pathways that reduce the regenerative capacity of axons, effective therapies have not yet emerged because removing the known inhibitory cues only partially restores regeneration, thus indicating the presence of additional inhibitory machineries that remain to be discovered (Song 2015).

Studies using model organisms such as Caenorhabditis elegans have begun to identify new genes important for axon regeneration, illustrating the power of the genetic approach. To identify more factors that control axon regeneration, a Drosophila sensory neuron injury model that exhibits class-specific axon regeneration was established and it was demonstrated that the class IV dendritic arborization (da) neuron is capable of regenerating its axon in the periphery but exhibits limited regrowth inside the CNS, resembling its mammalian counterpart at the phenotypic and molecular levels (Song, 2012). Using this model, a candidate-based genetic screen was performed focusing on axotomy-regulated genes from several organisms and Drosophila Rtca (CG4061), a cellular RNA-processing enzyme with unknown biological function, was identified as an inhibitor of CNS axon regeneration. Furthermore, it was found that Drosophila Archease, a RNA ligase cofactor, functions downstream of Rtca as a pro-regeneration factor. Rtca and Archease are components of the RNA repair and splicing pathway, and they regulate the unconventional mRNA splicing of Xbp1, a stress sensor. Thus, Xbp1 acts as a substrate, readout and downstream effector for the regulation of axon regeneration by the RNA repair and splicing pathway (Song 2015).

To assess axon regeneration, a previously described protocol was followed (Song, 2012). Briefly, using a two-photon laser, the axons of class IV da neurons (labeled with pickpocket (ppk)-CD4tdGFP) were severed in the ventral nerve cord (VNC) of second-instar larvae 48 h after egg laying (AEL); the degeneration of the remaining axons was confirmed after 1 d (72 h AEL) and their regeneration was assessed after 2 more days (120 h AEL). Using this model, the effect was measured of RtcaNP5057, an insertional loss of function (LOF) allele with a P-element inserted in the 5'-UTR, disrupting mRNA splicing and reducing transcript expression. Compared to wild types, which showed limited regrowth, new axons regrew extensively from the retracted axon stems and extended into the commissure region, forming elaborate branches and reconnected commissure segments in RtcaNP5057 larvae. Similar phenotypes were seen in transheterozygotes of RtcaNP5057 over a deficiency line, Df(1)BSC718, that lacks the Rtca locus and in a Rtca deletion allele, RtcaΔ, generated from imprecise excision of RtcaNP5057. Even stronger phenotypes were seen in RtcaΔmat, in which both the zygotic and maternal transcripts were removed. RtcaNP5057 is homozygous viable and fertile, so these larvae were derived from homozygous mutant mothers. The mothers of RtcaNP5057/Df(1)BSC718 transheterozygotes and RtcaΔ mutants were heterozygous for the wild-type allele and may provide maternal wild-type Rtca transcripts. The fact that RtcaΔmat mutants, in which both the zygotic and maternal transcripts were removed, showed a stronger phenotype than RtcaΔ zygotic mutants confirmed the maternal effect. Thus, the phenotype of RtcaNP5057 mutants compared to RtcaNP5057/Df(1)BSC718 transheterozygotes and RtcaΔ mutants is likely stronger because no wild-type maternal transcripts were provided to RtcaNP5057 mutants. The function of Drosophila Rtca is cell autonomous, as its RNA interference knockdown in class IV da neurons (ppk-Gal4>RtcaRNAi) but not in glial cells (repo-Gal4>RtcaRNAi) recapitulated the enhancement of regeneration. The regeneration phenotype was further quantified by assessing the following metrics, as described previously (Song, 2012): regeneration percentage, terminal branching and commissure regrowth. The enhancement of regeneration is unlikely to be due to developmental defects of axon outgrowth because, first, the overall axon patterning of class IV da neurons in the uncut VNC is grossly normal and second, reducing Rtca function in RtcaΔ mutants or transheterozygotes of RtcaNP5057 over Df(1)BSC718 or via Rtca RNAi in class IV da neurons did not result in obvious defects of axon terminal patterning in the VNC (Song 2015).

Tests were performed to see whether reducing Drosophila Rtca function would trigger a regenerative response in neurons normally incapable of regeneration by severing their axons in Rtca mutants. Indeed, Rtca removal in class III da neurons (labeled with 19-12-Gal4>CD4tdGFP, repo-Gal80), which unlike class IV da neurons did not regrow their axons that were severed in the periphery, elicited substantial regeneration in RtcaΔmat mutants and after RNAi knockdown of Rtca specifically in class III da neurons, leading to significant increases in the regeneration percentage, regeneration index and regeneration length (Song 2015).

Conversely, overexpression of Rtca in class IV da neurons (ppk-Gal4>Rtca) mildly reduced their regenerative potential in the peripheral nervous system (PNS). In wild-type class IV da neurons, which regenerated about 74% of the time, new axons extended beyond the lesion site and followed the axonal track. In contract, Rtca overexpression caused the incidence of regeneration to be reduced to 48% and the length of the new axons to be significantly shortened as well. These data indicate that Drosophila Rtca is an inhibitor of axon regeneration: not only does its removal cell-autonomously enhance axon regeneration in the CNS and enable regeneratively incompetent neurons such as class III da neurons to regrow their axons in the PNS, its overexpression in regeneratively competent neurons impedes axon regeneration in the periphery (Song 2015).

The expression pattern of Drosophila Rtca was examined via two approaches. First, the P-element inserted in Rtca 5'-UTR (RtcaNP5057) contains Gal4 in the same orientation as Rtca and thus can allow inference of Rtca expression via a UAS reporter. It was found that the Rtca-Gal4>CD4tdGFP reporter colocalized with the class IV da neuron marker ppk-CD4tdTomato, confirming its presence in class IV da neurons. Although Rtca-Gal4 expression was observed in other tissues in the PNS and VNC, the analyses indicate that Drosophila Rtca functions cell autonomously in neurons to inhibit axon regeneration (Song 2015).

A polyclonal antibody was generated against Drosophila Rtca. The protein was present in wild-type but not in RtcaΔmat null class IV and class III da neurons, and was enriched in the nucleus. Drosophila Rtca was also present in other types of multidendritic neurons (Song 2015).

To begin to understand the mechanisms underlying Drosophila Rtca's role in regeneration, attempts were made to determine how it genetically interacts with the known axon regeneration regulators Pten (phosphatase and tensin homolog) and the cytoskeletal regulator Rac1 GTPase. Deletion of Pten, a negative regulator of the mammalian target of rapamycin (mTOR) pathway, has been shown to increase CNS axon regeneration in both mammals and flies. Overexpression of Rtca in a Pten hypomorphic mutant background (PtenMGH6; ppk-Gal4>Rtca) or overexpression of Pten in Rtca null mutants (RtcaΔ; ppk-Gal4>Pten) largely abolished the enhancement of axon regeneration as seen in the VNC in PtenMGH6 or RtcaΔ mutants. This suggests that Rtca and Pten are likely to function in parallel pathways. Notably, double mutation of Rtca and Pten (RtcaNP5057; PtenMGH6) did not further improve regeneration, as compared to Rtca mutation alone, indicating the presence of additional brakes on regeneration. The regeneration phenotype in Rtca mutants appeared to be comparable to if not stronger than that in PtenMGH6 mutants or that seen with Akt overexpression (ppk-Gal4>Akt) (Song 2015).

Because Rac is required for regenerative axon outgrowth in C. elegans, this study overexpressed Rac1 in class IV da neurons (ppk-Gal4>Rac1). An increase was found in the number of axons initiating the regenerative response in the VNC but not in terminal branching or commissure regrowth; that is, there was a partial improvement in regeneration. Conversely, overexpressing a dominant negative (DN) form of Rac1 abolished the enhancement of CNS axon regeneration seen in Rtca null mutants (RtcaΔmat; ppk-Gal4>Rac1DN), whereas Rac1DN overexpression alone in class IV da neurons did not result in obvious axon regeneration defects in the PNS. It thus seems likely that Rac1 functions downstream of Rtca in a pathway that converges on regulation of the cytoskeleton (Song 2015).

Rtca is an RNA processing enzyme that possesses RNA-3'-phosphate cyclase activity and catalyzes the ATP-dependent conversion of a 3' phosphate to a 2',3'-cyclic phosphodiester at the end of RNA molecules (Genschik, 1997). The RNA 2',3'-cyclic phosphate ends are important in RNA metabolism -- for example, as intermediates during RNA repair by ligases (Popow, 2011; Remus, 2013). Rtcb (RNA 2',3'-cyclic phosphate and 5'-OH ligase) represents a new type of RNA ligase that joins 2',3'-cyclic phosphate and 5'-OH RNA ends to yield a 3'-5' phosphodiester splice junction. Specifically, Rtcb is known to possess cyclic phosphodiesterase activity, which hydrolyzes the 2',3'-cyclic phosphate to a 3'-phosphate, as well as ligase activity, which then joins the RNA 3'-phosphate to a 5'-OH RNA end. In addition, the specificity and efficacy of Rtcb's ligase activity can be enhanced by Archease (Desai, 2014; Popow, 2014), which is a small acidic protein conserved among Eukarya, Bacteria and Archaea. In Escherichia coli, RtcA and RtcB are encoded in a single operon, suggesting that they might cooperate to provide a healing and sealing function in an RNA repair pathway (Tanaka, 2011). In one scenario, healing would refer to the restoration of ligatable 2',3'-cyclic phosphate ends in the event of the inciting RNA damage directly generating RNA 3'-phosphates, or of the 2',3'-cyclic phosphate products of RNA transesterification being further processed to a 3'-phosphate by a 2',3'-cyclicphosphodiesterase. However, this model cannot readily be reconciled with the subsequent finding that RtcB readily joins 3'-phosphate to 5'-OH ends or 2',3'-cyclic phosphate to 5'-OH ends (Song 2015).

Therefore, the exact relationship between RtcA and RtcB remains undetermined. Notably, the RtcBA operon in E. coli is regulated by the σ54 coactivator RtcR, suggesting that the RNA repair functions are induced in response to cellular stress. Although the biological function of Rtca remains unknown, the enzyme is speculated to act in some aspect of cellular RNA processing . Taking into account these findings and the observation that loss of Rtca function enhances axon regeneration, it is hypothesized that the Rtca-Archease-dependent RNA repair and splicing pathway regulates axon regeneration. Specifically, it is speculated that axon injury triggers a type of cellular stress leading to RNA damage and splicing, producing RNA 3'-phosphates that need to be processed and rejoined by the Rtcb ligase, which is catalyzed by Archease. Because Rtca converts RNA 3'-phosphate to 2',3'-cyclic phosphate, it can slow the ligation process and impede regeneration. Consequently, silencing Rtca promotes axon regeneration. Following this reasoning, the role of Archease in axon regeneration was investigated (Song 2015).

To determine the role of the Drosophila Archease in axon regeneration, regeneration of class IV da neuron axons in the periphery was examined. To maximize the phenotype, the PNS axon injury protocol was modified as described previously (Song, 2012): axotomy was induced at 72 h AEL, degeneration was confirmed at 96 h AEL and regeneration was assayed at 120 h AEL. The Archease (CG6353) LOF mutant allele ArcheasePBc01013, which is an insertional allele with a P-element inserted into the 5'-UTR disrupting its mRNA splicing and eliminating Archease transcripts. Unlike in wild-type neurons, which exhibited substantial regrowth of their severed axons, axon regeneration was significantly impaired in ArcheasePBc01013 neurons, as revealed by a significant drop of the regeneration percentage, regeneration index and regeneration length. This phenotype was confirmed in transheterozygotes of ArcheasePBc01013 over either of the two deficiency lines, Df(3R)ED6076 or Df(3R)BSC678, that lack the Archease locus. The ArcheasePBc01013 mutation is larval lethal. ArcheasePBc01013 mutants and ArcheasePBc01013/DfED6076 and ArcheasePBc01013/DfBSC678 transheterozygotes showed similar phenotypes, suggesting that ArcheasePBc01013 is likely an amorphic allele. The function of Archease is required cell-autonomously, as class IV da neuron-specific knockdown of Archease (ppk-Gal4>ArcheaseRNAi) but not glial cell knockdown (repo-Gal4>ArcheaseRNAi) was sufficient to phenocopy the regeneration failure. Moreover, loss of function of both Rtca and Archease (RtcaNP5057; ArcheasePBc01013) completely abolished the axon regeneration-promoting effect in the VNC seen in RtcaNP5057 mutants, producing many retracted or stalled axon stems. This epistasis analysis indicates that Archease is a pro-regeneration factor downstream of Rtca and that they act in opposing ways to regulate axon regeneration (Song 2015).

What might be the RNA substrates processed by this Drosophila Rtca-Archease-dependent RNA repair and splicing pathway for the regulation of axon regeneration? This study investigated X-box binding protein 1 (Xbp1) as a candidate substrate for three reasons. First, cellular stress such as endoplasmic reticulum (ER) stress triggers an adaptive intracellular signaling cascade known as the unfolded protein response (UPR). One main branch of the UPR is the activation of Ire1, which cleaves Xbp1 pre-mRNA in the cytoplasm, converting the unspliced Xbp1μ, a putative transcriptional repressor, into the unconventionally spliced Xbp1s by eliminating an intron (26 nucleotides in mammals, 23 in flies) that changes the open reading frame of the third exon, resulting in a new protein that acts as a transcriptional activator (Yoshida, 2001). Xbp1s directly activates ER stress target genes to facilitate refolding and also degradation of misfolded proteins (Ron, 2007). Second, the RNA ligase Rtcb and its cofactor Archease are involved in the unconventional splicing induced by the UPR, and Archease is required for the splicing of the Xbp1 mRNA (Jurkin, 2014). Third, loss of xbp1 function in C. elegans results in severely reduced axon regeneration (Nix, 2014). To determine the function of Xbp1 in axon regeneration in the PNS and CNS, a mutant allele, Xbp1k13803, was used that has a P-element inserted into its 5'-UTR, thus reducing transcripts (Ryoo, 2007a). Axon regeneration in the periphery was mildly reduced in these mutants. This defect was stronger in transheterozygotes of Xbp1k13803 over a deficiency line, Df(2R)BSC484, that lacks the Xbp1 locus, suggesting that Xbp1k13803 is likely a hypomorphic allele. Class IV da neuron-specific (ppk-Gal4>Xbp1RNAi) but not glia-specific (repo-Gal4>Xbp1RNAi) RNAi of Xbp1 reproduced the impairment of regeneration, indicating it functions cell-autonomously. Moreover, double mutation of Rtca and Xbp1 (RtcaNP5057; Xbp1k13803) dampened the enhancement of CNS axon regeneration seen in RtcaNP5057 mutants, indicating that Xbp1 is indeed a pro-regeneration factor downstream of Drosophila Rtca. Consistent with these lines of evidence, overexpression of the spliced form Xbp1s in class IV da neurons significantly enhanced axon regeneration in the VNC, and it also promoted axon regeneration in the periphery when overexpressed in class III da neurons. The observations that Rtca; Xbp1 double mutation did not completely eliminate the enhanced regeneration phenotype in Rtca mutants and that Xbp1s overexpression led to milder enhancement of regeneration as compared to Rtca LOF, suggest that additional substrates contribute to regeneration regulation (Song 2015).

To directly assess the nonconventional splicing of Xbp1 mRNA in vivo, a heat-shock model was used. Fly larvae of various genotypes underwent a 40°C heat shock and the abundance of Xbp1 splice variants was assessed using semiquantitative RT-PCR. The Xbp1s/Xbp1μ ratio was then quantified. Heat-shock induced the expression of the spliced form, Xbp1s. In contrast to the enhanced expression of Xbp1s in Rtca mutants, Xbp1s levels were greatly reduced in Archease LOF mutants. Double mutants of Rtca and Archease resembled Archease mutants in the reduction of Xbp1 splicing. Taken together, these data indicate that Drosophila Rtca and Archease in the RNA repair and splicing pathway negatively and positively regulate the stress-induced Xbp1 mRNA splicing, respectively, so that Xbp1 acts as a readout and effector for the regulation of axon regeneration (Song 2015).

Having established the role of Rtca in axon regeneration in Drosophila, the study went on to determine whether its function is evolutionarily conserved in mammals. The expression pattern of the mammalian ortholog of Drosophila Rtca was examined in vitro. Antibodies raised against human RTCA recognized rat Rtca in the cell bodies and processes of cultured hippocampal neurons. Moreover, the expression of Rtca transcripts in the dorsal root ganglion (DRG) in vivo increased progressively throughout development, reaching the highest level in adults. Using quantitative RT-PCR, it was found that Rtca transcript levels in the DRG were significantly reduced following lesion of the sciatic nerve peripherally, but not lesion of the central axon branch of DRG neurons with a spinal cord hemisection. Since the peripheral processes of DRG neurons are capable of regeneration, whereas their central axons that project into the spinal cord fail to regrow after injury, the selective suppression of Rtca following peripheral injury supports the hypothesis that the persisting expression of Rtca is inhibitory to axon regeneration in the CNS. Furthermore, in agreement with the overexpression phenotype in flies, overexpression of Rtca in cultured hippocampal neurons reduced axon complexity and markedly reduced proximal axonal branching without affecting total axon length, indicating an inhibitory function of Rtca (Song 2015).

It was next asked whether knocking out Rtca during development enhances axon regeneration of adult mouse retinal ganglion cells (RGCs) in vivo. For this purpose, a mutant allele was generated with a lacZ cassette inserted after the third exon to disrupt splicing and reduce transcription (to ~19%), thereby generating RtcalacZ_loxP (RtcaIns/Ins) mutant mice. Rtca protein level was also reduced to ~18% in the mutants, suggesting this is a hypomorphic allele. Homozygous RtcaIns/Ins mice were born, although at less than the Mendelian ratio. By adulthood, there were no obvious differences in RGC number or RGC axon morphology among mutant (RtcaIns/Ins), heterozygous (RtcaIns/+) and wild-type (Rtca+/+) animals. Since the lacZ cassette is inserted into the Rtca locus, it can be used as a reporter for examining Rtca expression. β-Galactosidase staining was observed in the RGC layer of RtcaIns/+ mice but not in Rtca+/+ littermates . Moreover, β-galactosidase immunostaining in RtcaIns/+ mice showed distinct expression of the lacZ reporter in NeuN+ neurons in the retina, which was absent in Rtca+/+ littermates, indicating that Rtca is indeed expressed in RGCs. To assess RGC axon regeneration, optic nerve crush was perfored in RtcaIns/Ins, RtcaIns/+ and Rtca+/+ littermate mice at two developmental time points, postnatal day (P) 35 and 2-3 months old, and the extent of axon regeneration was measured in the optic nerve after 2 weeks or 3 weeks, respectively. RtcaIns/+ and Rtca+/+ mice injured at P35 did not exhibit substantial axon regrowth beyond the crush site, whereas RtcaIns/Ins mutant mice showed a substantial increase in the number of regenerating axons at various distances from the injury site, with some regenerating axons extending over 1.5 mm beyond the crush site. Mice operated on at 2-3 months old showed a milder regeneration enhancement phenotype. In these animals, curving, turning and looping of axons were observed, indicative of new axon growth, and the furthest distance that axons traveled beyond the injury site was about 3.5 times longer in the mutants. The crush site was further marked by the presence of ED1 staining, which labels infiltrating macrophages. Whereas axons rarely penetrated beyond the ED1+ region in sibling controls, a large number of axons were seen hundreds of microns beyond the ED1+ region in RtcaIns/Ins mutants. Reducing Rtca function did not affect RGC survival after injury, confirming that this increase in regenerating axons was not secondary to an increase in RGC numbers. The finding that reducing Rtca expression increased the regenerative potential of adult RGCs thus provides evidence for a potentially conserved role of Rtca as an anti-regeneration factor (Song 2015).

These findings reveal an important role of the RNA repair and splicing pathway in regulating the intrinsic axon regeneration potential in response to PNS and CNS injury in Drosophila. Rtca and Archease integrate the injury signals triggered by axotomy and lead to the activation of downstream effectors such as the stress response cascade involving Xbp1 splicing, affecting the ability of a neuron to regenerate. Axon injury has been suggested as a cellular stress, and the mTOR pathway, a potential determinant of neuronal regeneration competence, could be inactivated under stress conditions such as hypoxia or DNA damage (Lu, 2014). Notably, Xbp1 splicing has been observed in RGCs after optic nerve injury and forced activation of Xbp1 promotes RGC survival (Hu, 2014). This work implicates proper splicing of Xbp1 as also important for axon regeneration in Drosophila (Song 2015).

Moreover, recent work in C. elegans also suggests the involvement of stress response pathways, such as heat-shock, hypoxia and UPR, in axon regeneration (Nix, 2014). However, how the injury signal is relayed to the stress response is unclear. This work identifies a missing link and implicates the Rtca-Archease-dependent RNA metabolism machinery as a regeneration regulator. A priori, axonal injury could either signal directly to the stress pathways, which then recruit Rtca-Archease, or alternatively, Rtca and Archease may represent injury response elements upstream of the stress pathways. The results showed that the Xbp1-dependent UPR pathway acts downstream of Rtca-Archease in controlling axon regeneration, and the remaining question is whether and how it impinges on other stress pathways, such as hypoxia or DNA damage. It will be important in future studies to identify other substrates, in addition to Xbp1, that are modified by Rtca-Archease, and to search for response genes downstream of Xbp1. This study raises the prospect of manipulating Rtca, Archease and Xbp1 as potential therapeutic interventions for treating nervous system injury (Song 2015).

As a first step to determining whether the Rtca pathway may have an evolutionarily conserved function in axon regeneration, this study has examined CNS axon regeneration after optic nerve crush in a hypomorphic mouse mutant allele of Rtca and evidence was obtained suggesting that this is indeed the case. The enhancement of RGC axon regeneration phenotype in the Rtca mutant is modest as compared to that seen in Pten, Klf4 or Socs3 knockouts. This may be due to the residual Rtca function in this hypomorphic allele or to developmental compensation. Future experiments using mammalian injury models to examine the Rtca null allele and to assess other components of the RNA repair and splicing pathway are therefore warranted to further define its potential role in axon regeneration (Song 2015).

The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER) stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions [the hydrophobic region 2 (HR2) and the C-terminal translational pausing site] present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. This study generated a Drosophila Xbp1 deletion mutant (Excision101) lacking both HR2 and C-terminal region, but not the Ire1 splicing site. Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. The results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced (Coelho, 2014. PubMed).

Xbp1-independent ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila

The unfolded protein response (UPR) is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Inositol-requiring enzyme-1 (Ire1)
signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. This study shows that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD), including for the Fatty acid transport protein (Fatp). Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. These results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm (Coelho, 2013).

The endoplasmic reticulum (ER) is the cell organelle where
secretory and membrane proteins are synthesized and folded.
When the folding capacity of the ER is impaired, the presence
of incorrectly folded (misfolded) proteins in the ER causes ER
stress and activates the unfolded protein response (UPR), which
helps to restore homeostasis in the ER. In higher eukaryotes, the activation of
the UPR is accomplished via three signaling pathways induced
by ER-resident molecular ER stress sensors: protein kinase
(PKR)-like ER kinase (PERK),activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (Ire1).
Being conserved in all eukaryotes, Ire1 contains an ER luminal
domain, which is involved in the recognition of misfolded
proteins, and
cytoplasmic endoribonuclease and kinase domains, which are
involved in the activation of downstream pathways. Activated
Ire1 mediates the nonconventional splicing of an intron from X
box binding protein 1 (Xbp1) mRNA (or HAC1 mRNA, the yeast
Xbp1 ortholog), causing a frameshift during translation, thereby
introducing a different carboxyl domain in the Xbp1 protein. Xbp1spliced is an effective
transcription factor that regulates the expression of ER chaperones
and other target genes (Coelho, 2013).

In addition to mediating Xbp1 mRNA splicing, cell culture
studies demonstrated that Ire1 promotes the degradation of
mRNAs encoding ER-targeted proteins, a process called
RIDD (regulated Ire1-dependent decay), to reduce the load of
ER client proteins during ER stress. The cytosolic domain
of mammalian IRE1 binds Traf2 (tumor necrosis factor receptor-associated
factor 2), an upstream activator of
the c-Jun N-terminal kinase (JNK) signaling pathway. This IRE1/
Traf2 interaction is also independent of Xbp1 splicing and may
lead to the activation of apoptosis after prolonged ER stress
(Coelho, 2013 and references therein).

In the Drosophila photoreceptor cells, the rhabdomere is the
light-sensing organelle, a stack of photosensitive apical microvilli
that is formed during the second half of pupal development. The rhabdomere
is formed in the apical domain of each photoreceptor
cell, which after a 90 rotation extends its apical domain along
the proximal-distal axis of the retina. The growth of the rhabdomere
requires the delivery of large amounts of membrane and
proteins into this structure, imposing a considerable demand
to the cellular mechanisms controlling protein folding and membrane
production in the ER (Coelho, 2013).

Among the proteins targeted to the developing rhabdomeres
are the rhodopsins, the light-sensitive proteins, and
other proteins involved in the transduction of the light stimuli.
Rhodopsin-1 (Rh1) is a seven transmembrane domain protein
that starts to be expressed by 78% of pupal life and is delivered to the rhabdomeres of the outer
photoreceptors (R1-R6), in a trafficking process that requires
the activity of Rab11, MyosinV, and dRip11. The delivery of Rh1 to the rhabdomere is
required for rhabdomere morphogenesis because in Rh1-null
mutants, the rhabdomere does not form, causing degeneration
of the photoreceptors (Coelho, 2013).

In mammalians, the microRNA mir-708 is upregulated by CCAAT enhancer-binding
protein homologous protein (CHOP) to control rhodopsin expression levels and prevent an
excessive rhodopsin load into the ER .
In Drosophila, Ire1 signaling is activated in the photoreceptors
upon expression of Rh1 folding mutants or in ninaA
mutations that cause the accumulation of misfolded Rh1
in the ER. However, the role of Ire1
signaling during normal photoreceptor differentiation remains
unknown. This study shows that Ire1 signaling is activated
in the photoreceptors during pupal stages of Drosophila
development. Ire1 mutant photoreceptors have defects in
the delivery of Rh1 to the rhabdomere and the secretion of
Spacemaker/Eyes Shut (Spam/Eys) into the interrhabdomeral
space (IRS). Surprisingly, Xbp1-null mutant photoreceptors
have a milder phenotype with no defects in Rh1 delivery into
the rhabdomere or Spam/Eys secretion. Targets of RIDD are
upregulated in Ire1 mutant retinas, including the fatty acid
transport protein (fatp), a known regulator of Rh1 protein levels. Finally, it was shown that the regulation of
fatp levels by RIDD is critical for normal Rh1 delivery into the
rhabdomere (Coelho, 2013).

Studies in mammalian systems revealed that the Ire1/Xbp1
signaling pathway is important during development for the differentiation
of secretory cells. For example, Xbp1 'knockout' mice
have defects in the differentiation of antibody-secreting plasma
cells and secretory cells of the exocrine
glands of the pancreas. Presumably, in these
cases, activation of Ire1/Xbp1 signaling is required to increase
the capacity of the ER to fold and process the high load of
secreted proteins (Coelho, 2013).

The present results demonstrate that Ire1 signaling is required
for photoreceptor differentiation and rhabdomere morphogenesis,
a process that also imposes a high demand to the capacity
of the ER to fold proteins such as Spam/Eys and Rh1. As shown,
Ire1 mutant photoreceptors have defects in the secretion of
Spam/Eys to the IRS and in the delivery of Rh1 to the rhabdomere.
However, activation is seen of the Xbp1-EGFP reporter
starting at 48 hr of pupal development, well before when the
Spam/Eys secretion and Rh1 delivery defects are observed. Presumably,
the folding of other unidentified proteins during these
earlier stages might also require Ire1 signaling. It is noteworthy
though, that in mutant B lymphocytes modified to lack antibody
production, Ire1 is still activated (and Xbp1 spliced) upon
lymphocyte differentiation to plasma cells.
Activation of Ire1/Xbp1 signaling in this context seems to be
part of the process of plasma cell differentiation, independently
of the accumulation of misfolded proteins in the ER lumen (Coelho, 2013).

Ire1 function is also required for the regulation of the membrane
lipids. In mammalians, Ire1/Xbp1 signaling regulates the
biosynthesis of phospholipids and other lipids. A study in yeast demonstrated that Ire1
is activated by 'membrane aberrancy,' a condition of stress
caused by the experimental depletion of inositol. Activation of Ire1 in this case occurs by a mechanism
that is distinct from the one involving the recognition of
misfolded proteins by the luminal domain of Ire1.
Furthermore, Ire1 can be activated by direct binding
of flavonoids, such as quercetin, to a pocket present in the cytoplasmic
domain of Ire1, in a mechanism that is also independent
of the binding of misfolded proteins to Ire1. The present results do not clarify if Ire1 activation in the
photoreceptors during pupal stages results from the accumulation
of misfolded proteins in the ER lumen or an imbalance in the
membrane lipids (Coelho, 2013).

The results demonstrate that Ire1 signaling is required for
photoreceptor differentiation and rhabdomere morphogenesis
in an Xbp1-independent manner. Studies using cell culture paradigms
demonstrated that, in addition to mediating Xbp1 mRNA
splicing, Ire1 also promotes RIDD, the degradation of mRNAs
encoding ER-targeted proteins, but the physiological significance
of the RIDD mechanism is unknown. Quantitative
RT-PCR results show that RIDD targets are upregulated in Ire1
mutant eyes, including fatp, a regulator of Rh1 protein levels. The results show that regulation of fatp
mRNA by RIDD is critical for rhabdomere morphogenesis
because the experimental downregulation of fatp mRNA by
RNAi rescues the Rh1 rhabdomere delivery defect observed in
Ire1 mutants (Coelho, 2013).

Rh1 protein levels and Rh1 delivery to the rhabdomere are very
sensitive to the levels of sphingolipids
and phosphatidic acid. Increased fatp levels
may lead to an increase in the levels of fatty acids and, subsequently,
phosphatidic acid, which is known to downregulate
Rh1 protein levels and cause rhabdomere morphogenesis
defects. High levels of phosphatidic acid
disrupt the Arf1-dependent transport of membrane to the developing
rhabdomere. The results show that
phosphatidic acid levels are elevated in Ire1 mutant retinas,
and lowering phosphatidic acid levels by expression of LPP rescues
the defects observed in Ire1 mutants, demonstrating that
Ire1/fatp-dependent regulation of fatty and phosphatidic acids
levels is important for rhabdomere morphogenesis in Drosophila.
In addition, it is possible that the increase in phosphatidic acid
levels in Ire1 mutant photoreceptors is also caused by the
activation of PERK because upregulation was observed of the
PERK pathway mediator ATF4 in Ire1 mutant photoreceptors, and in cell culture models, it was shown that
PERK is able to phosphorylate diacylglycerol and generate
phosphatidic acid.
In conclusion, the results, using well-characterized genetic
tools (Ire1 and Xbp1-null mutations) and a developmental paradigm
(photoreceptor differentiation in the Drosophila pupa),
demonstrate the physiological relevance of Xbp1-independent
mechanisms downstream of Ire1 signaling (Coelho, 2013).

Alterations in the quality, quantity and architecture of baseline and recovery sleep have been shown to occur during aging. Sleep deprivation induces endoplasmic reticular (ER) stress and upregulates a protective signaling pathway termed the unfolded protein response (UPR). The effectiveness of the adaptive UPR is diminished by age. Previously, it has been shown that endogenous chaperone levels alter recovery sleep in Drosophila melanogaster. This study reports that acute administration of the chemical chaperone sodium 4-phenylbutyrate (PBA) reduces ER stress and ameliorates age-associated sleep changes in Drosophila. PBA consolidates both baseline and recovery sleep in aging flies. The behavioral modifications of PBA are linked to its suppression of ER stress. PBA decreases splicing of X-box binding protein 1 (XBP1) and upregulation of phosphorylated elongation initiation factor 2α (p-eIF2α), in flies that were subjected to sleep deprivation. It was also demonstrated that directly activating ER stress in young flies fragments baseline sleep and alters recovery sleep. Alleviating prolonged/sustained ER stress during aging contributes to sleep consolidation and improves recovery sleep/ sleep debt discharge (Brown, 2014).

With advancing age, humans demonstrate a number of sleep disruptions, which contribute to poor health consequences and accelerated senescence. The mechanisms that lead to these negative health outcomes, however, remain unclear. This study shows that both baseline and recovery sleep change with age and these alterations can be modulated by the chemical chaperone PBA. PBA is a low molecular weight fatty acid that has been shown to act as a chemical chaperone for misfolded or mislocalized proteins. It has also been shown to suppress ER-stress induced cell death without the aid of endogenous ER chaperones. It was also demonstrated in this study that directly inducing ER stress in young flies fragments baseline sleep and alters recovery sleep, phenocopying aged flies. Sleep fragmentation may contribute to accelerated aging and it has been suggested that an intervention that consolidates sleep may delay the aging process and increase life span (Brown, 2014).

Aged flies exhibit less total baseline sleep and an altered homeostatic response in that they recover sleep over an extended 12 h period, whereas young flies recoup lost sleep within a much shorter window. Similar sleep disturbances are seen in humans and mammals, where aged individuals lose the ability to initiate and maintain nighttime sleep and recovery sleep after prolonged wakefulness is altered. Similar to this study, a significant age by time interaction was demonstrated, where young flies recover sleep in the first 4-5 h following sleep deprivation. Sleep deprived aged flies however, recover sleep over much a longer period. Variations in behavioral results compared with those existing in the literature are possibly due to this study's use of video analysis, which has been shown to be more accurate than DAMS in measurements of fly sleep, in particular daytime sleep and architecture (Brown, 2014).

During the course of this study, an increase in locomotor activity in both young and aged flies treated with PBA was observed. A previous study found that PBA maintains locomotor vigor (activity) during aging in Drosophila. These results suggest that PBA is in fact increasing sleep and not merely reducing movement in the aged flies. Although total wake did not change in the young flies treated with PBA, wake became more consolidated. Not observing changes in total wake is possibly due to a ceiling effect in the young healthy flies. These results indicate that there is an age by drug interaction in the modification of sleep by PBA that remains to be elucidated (Brown, 2014).

Aging also impairs quality control systems that are necessary for protein homeostasis. Earlier work has demonstrated that aging decreases BiP levels and increases pro-apoptotic factors such as CCAAT/enhancer-binding protein-homologous protein (CHOP) in the cerebral cortex of mice. Young flies experience less ER stress and their adaptive UPR is fully functional. Aged flies, on the other hand, are under chronic ER stress conditions with a diminished adaptive UPR. Therefore, basal ER stresses for the two groups are vastly different and likely contributes to the disparate responses to sleep deprivation. The young flies are readily able to handle acute sleep deprivation, whereas aged flies, already under stress, are overwhelmed by the secondary stressor. Addition of PBA has little effect on recovery sleep in young flies; however, PBA treatment ameliorates existing ER stress and suppresses further UPR induction in aged flies. As a result, treated aged flies display more efficient recovery sleep or are able to discharge sleep debt more efficiently than untreated aged flies (Brown, 2014).

Although no significant differences were found in BiP expression in flies treated with PBA, XBP1 was significantly affected by PBA treatment. PBA not only reduces the amount of spliced XBP1, it also reduces the expression of the unspliced variant as well. PBA likely suppresses UPR induction by chaperoning unfolded proteins, so that BiP stays bound to IRE1 and PERK and delays or prevents its activation, even under the condition of sleep deprivation. Phospho-IF2α levels are significantly reduced in the PBA treated aged flies subjected to sleep deprivation. Changes in p-eIF2α can be directly linked to the behavioral changes in the aged flies given that increased p-eIF2α facilitates baseline and recovery sleep. The reduced eIF2α phosphorylation in PBA treated animals may account for the improved homeostatic response in the aged SD flies (Brown, 2014).

This study also chemically induced ER stress in young flies and then subjected them to sleep deprivation. This acute tunicamycin treatment was found to decrease and fragment baseline sleep and phenocopy the recovery sleep behavior that is seen in the aged flies. There are two possible explanations for the tunicamycin results. First, tunicamycin activity leads to inhibition of glycosylation and general misfolding of proteins, which will ultimately result in ER stress. Secondly, tunicamycin could potentially prevent maturation of glycoproteins required for sleep maintenance. This data demonstrates that ER stress induces sleep fragmentation. Together with previous findings that sleep deprivation caused ER stress and activated the UPR, these results suggests that both processes are intimately linked and feedback on one another (Brown, 2014).

PBA has been shown to significantly increase life span in Drosophila, purportedly through inhibition of HDAC activity. The moderate effects of PBA on sleep consolidation in young sssp1 mutant flies suggest that protein stabilization by PBA is only partially ameliorating defects in these animals. The decrease in sleep in the ctrl sss background strain treated with PBA supports the increase and consolidation of wake in the wild-type lines. It has been previously shown that induction of ER stress impairs waking in mice. The study speculates that application of PBA also promotes wake-active neuronal function and consolidates wake in the flies by reducing ER stress. The issue is not the accumulation of sleep debt, but the discharge of sleep debt that is impaired in the aged flies. By ameliorating ER stress and suppressing activation of the UPR, PBA allows for more efficient recovery sleep in the aged flies. Since PBA ameliorates ER stress by directly reducing aberrant protein load, treating Drosophila with PBA supplements chaperone activity and decreases the burden of misfolded proteins that occurs as a consequence of an external stressor, sleep deprivation, as well as normal aging (Brown, 2014).

Mitochondrial dynamism (fusion and fission) is responsible for remodeling interconnected mitochondrial networks in some cell types. Adult cardiac myocytes lack mitochondrial networks, and their mitochondria are inherently 'fragmented'. Mitochondrial fusion/fission is so infrequent in cardiomyocytes as to not be observable under normal conditions, suggesting that mitochondrial dynamism may be dispensable in this cell type. However, it has been previously reported that cardiomyocyte-specific genetic suppression of mitochondrial fusion factors Optic atrophy 1 (Opa1) and Mitofusin (Marf) evokes cardiomyopathy in Drosophila hearts. Fusion-mediated remodeling of mitochondria may be critical for cardiac homeostasis, although never directly observed. Alternately, inner membrane Opa1 and outer membrane mitofusin/MARF might have other as-yet poorly described roles that affect mitochondrial and cardiac function. This study compared heart tube function in three models of mitochondrial fragmentation in Drosophila cardiomyocytes: Drp1 expression, Opa1 RNAi, and mitofusin MARF RNA1. Mitochondrial fragmentation evoked by enhanced Drp1-mediated fission did not adversely impact heart tube function. In contrast, RNAi-mediated suppression of either Opa1 or mitofusin/MARF induced cardiac dysfunction associated with mitochondrial depolarization and ROS production. Inhibiting ROS by overexpressing superoxide dismutase (SOD) or suppressing ROMO1 prevented mitochondrial and heart tube dysfunction provoked by Opa1 RNAi, but not by mitofusin/MARF RNAi. In contrast, enhancing the ability of endoplasmic/sarcoplasmic reticulum to handle stress by expressing Xbp1 rescued the cardiomyopathy of mitofusin/MARF insufficiency without improving that caused by Opa1 deficiency. The study concludes that decreased mitochondrial size is not inherently detrimental to cardiomyocytes. Rather, preservation of mitochondrial function by Opa1 located on the inner mitochondrial membrane, and prevention of ER stress by mitofusin/MARF located on the outer mitochondrial membrane, are central functions of these 'mitochondrial' fusion proteins
(Bhandari, 2015).

By performing a side-by-side detailed comparison of cardiomyopathies provoked by interrupting fusion of either the outer or inner mitochondrial membranes, this study identified distinct cellular mechanisms for the different molecular lesions. RNAi-mediated suppression of outer mitochondrial membrane mitofusin/MARF and inner mitochondrial membrane Opa1 provokes similar overt phenotypes: in both models mitochondrial size is approximately halved, the proportion of depolarized (sick) mitochondria increases to ~40%, mitochondrial ROS production is comparably greater (~50%), and the heart tubes exhibit similar reductions in fractional shortening. However, the cardiac defect caused by Opa1 deficiency is readily corrected by attacking the disease at the level of mitochondrial ROS production, through SOD expression or ROMO1 suppression. Indeed, mitochondrial structural and functional abnormalities are also improved by these genetic maneuvers, suggesting that they interrupt a vicious cycle of ROS-induced mitochondrial degeneration provoked by Opa1 insufficiency. Thus, interrupting endogenous mitochondrial ROS production greatly abrogates both the mitotoxicity and the cytotoxicity evoked by Opa1 suppression. This suggests a central role for mitochondrial degeneration in the Opa1-deficient fly heart model and, by extension, other heart diseases caused by defective inner mitochondrial membrane fusion proteins (Bhandari, 2015).

Whereas mitochondrial size and polarization status are similarly impaired in mitofusin/MARF insufficient heart tubes, neither of the mitochondrial-targeted interventions directed at reducing ROS, both of which rescue Opa1 deficient hearts, improve the cardiomyopathy induced by mitofusin/MARF deficiency. Indeed, whereas transgenic expression of SOD1 or SOD2 normalizes both ROS and heart tube function in Parkin-deficient heart tubes and Opa1-deficient heart tubes, it is remarkable that SOD fails to improve ROS levels in the mitofusin/MARF deficient heart tubes. Together with the original observation of transient heart tube functional improvement with SOD1, these observations suggest that there is 'ROS escape' in the mitochondrial fusion impaired model that confers resistance to SOD expression or ROMO1 suppression. Instead, heart tube function is normalized without improving either mitochondrial structural or functional abnormalities by genetically enhancing the cardiomyocytes' ability to handle ER stress through XBP1 expression. These results, although surprising, are consistent with an essential role for mitofusin-mediated mitochondrial-ER/SR cross-talk in managing the ER stress response as proposed earlier in heart and in other tissues (Bhandari, 2015).

Previously, the consequences of Opa1 deficiency on Drosophila eye phenotypes have been found to be rescuable with SOD1, which is in accordance with findings of this study. Another study in Drosophila neurons and skeletal muscle also supports an important role for mitofusins, but not Opa1, as modulators of ER stress. However, only human Mfn2, and not human Mfn1, can correct abnormalities induced by mitofusin/MARF suppression in flies. This contrasts with findings in this study that cardiac-specific expression of either human Mfn1 or Mfn2 will fully correct cardiomyopathy induced by cardiomyocyte-specific MARF RNAi. Furthermore, ER dysmorphology has been found in MARF-deficient fly tissues, which was not detected in MARF-deficient heart tubes. It is likely either that the heart has different requirements for mitofusins and ER/SR morphology, or that the powerful tinman gene promoter used for cardiomyocyte-specific gene manipulation confers different expression characteristics to the heart models. Either way, the overall conclusions regarding a role of ER stress in mitofusin deficiency are in agreement (Bhandari, 2015).

Cardiomyocyte mitochondria are the Oompa-Loompas of the heart (with apologies to Roald Dahl): They are diminutive, structurally homogenous, and frequently overlooked despite toiling endlessly behind the scenes to keep the place running. Research has tended to focus on the mitochondrial work product (cardiac metabolism) and the means by which the general mitochondrial population is sustained (through biogenesis), rather than the fate of individual organelles. Indeed, individual cardiomyocyte mitochondria seem hardly worthy of observation, being monotonously similar in appearance and lacking the morphometric remodeling or intra-cellular mobility that has sparked detailed investigations (and visually engaging movie clips) in other cell types. The data presented in this study emphasize that (for mitochondria as well as Oompa-Loompas) size is not the critical determinant of function; it is literally what is inside that counts. Accordingly, one should eschew generalizations and extrapolations of mitochondrial status and dysfunction based strictly on morphometry (Bhandari, 2015).

The pathogenic human Torsin A in Drosophila activates the unfolded protein response and increases susceptibility to oxidative stress

Dystonia1 (DYT1) dystonia is caused by a glutamic acid deletion (ΔE) mutation in the gene encoding Torsin A in humans (HTorA). To investigate the unknown molecular and cellular mechanisms underlying DYT1 dystonia, this study performed an unbiased proteomic analysis. It was found that the amount of proteins and transcripts of an Endoplasmic reticulum (ER) resident chaperone Heat shock protein cognate 3 (HSC3) and a mitochondria chaperone Heat Shock Protein 22 (HSP22) are significantly increased in the HTorAΔE– expressing brains compared to the normal HTorA (HTorAWT) expressing brains. The physiological consequences include an increased susceptibility to oxidative and ER stress compared to normal HTorAWT flies. The alteration of transcripts of Inositol-requiring enzyme-1 (IRE1)-dependent spliced X box binding protein 1 (Xbp1), several ER chaperones, a nucleotide exchange factor, Autophagy related protein 8b (ATG8b) and components of the ER associated degradation (ERAD) pathway and increased expression of the Xbp1-enhanced Green Fluorescence Protein (eGFP) in HTorAΔE brains strongly indicate the activation of the unfolded protein response (UPR). In addition, perturbed expression of the UPR sensors and inducers in the HTorAΔE Drosophila brains results in a significantly reduced life span of the flies. Furthermore, the types and quantities of proteins present in the anti-HSC3 positive microsomes in the HTorAΔE brains are different from those of the HTorAWT brains. Taken together, these data show that HTorAΔE in Drosophila brains may activate the UPR and increase the expression of HSP22 to compensate for the toxic effects caused by HTorAΔE in the brains (Kim, 2015).

Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the lumen of the ER. A cell responds to ER stress with the unfolded protein response (UPR), a complex program of transcriptional and translational changes aimed at clearing misfolded proteins. Secretory tissues and cells are particularly well adapted to respond to ER stress because their function requires high protein production and secretory load. The insect male accessory gland (AG) is a secretory tissue involved in male fertility. The AG secretes many seminal fluid proteins (SFPs) essential for male reproduction. Among adult Drosophila tissues, this study finds that genes upregulated by ER stress are most highly expressed in the AG, suggesting that the AG is already undergoing high levels of ER stress due to its normal secretory functions. It was hypothesized that induction of excessive ER stress in the AG above basal levels, would perturb normal function and provide a genetic tool for studying AG and SFP biology. To test this, excessive ER stress was they genetically induced in the AG by conditional 1) expression of a misfolded protein or 2) knockdown of the UPR regulatory protein, BiP (Heat shock 70-kDa protein cognate 3). Both genetic manipulations induced excessive ER stress in the AG, as indicated by the increase in Xbp1 splicing, a marker of ER stress. Both models resulted in a large decrease in or loss of SFP production and male infertility. Sperm production, motility, and transfer appeared unaffected. The induction of strong ER stress in the insect male AG may provide a simple way for studying or manipulating male fertility, as it eliminates AG function while preserving sperm production (Chow, 2015).

Expression of genes in the endoplasmic reticulum (ER) beyond its protein folding capacity activates signaling pathways that are collectively referred to as the Unfolded Protein Response (UPR). A major branch of the UPR pathway is mediated by IRE1, an ER-tethered endonuclease. Upon ER stress-induced activation, IRE1 splices the mRNA of XBP1, thereby generating an active isoform of this transcription factor. During normal Drosophila development, tissues with high protein secretory load show signs of IRE1/XBP1 activity indicative of inherent ER stress associated with those cell types. This study reports that the XBP1 promoter activity itself is enhanced in secretory tissues of Drosophila, and it can be induced by excessive ER stress. Specifically, the study developed a Drosophila XBP1 transcription reporter by placing dsRed under the control of the XBP1 intergenic sequence. DsRed expression in these xbp1p>dsRed transgenic flies shows patterns similar to that of xbp1 transcript distribution. In healthy developing flies, the reporter expression is highest in salivary glands and the intestine. In the adult, the male reproductive organs show high levels of dsRed. These tissues are known to have high protein secretory load. Consistently, the xbp1p>dsRed reporter is induced by excessive ER stress caused by mutant Rhodopsin-1 overexpression. These results suggest that secretory cells suffer from inherent ER stress, and the xbp1p>dsRed flies provide a useful tool in studying the function and homeostasis of those cells (Ryoo, 2013).

A modified UPR stress sensing system reveals a novel tissue distribution of IRE1/XBP1 activity during normal Drosophila development

Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor protein, IRE1, ATF6, and PERK, respectively. In Drosophila, IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, previously reported XBP1 stress sensing systems (Souid, 2007; Ryoo, 2007a) were modified, based on the recent reports regarding the unconventional splicing of XBP1/HAC1 mRNA (Aragon, 2009; Yanagitani, 2009; Yanagitani, 2011). The improved XBP1 stress sensing system allowed detection of new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide novel insights into the brain homeostasis regulated by the UPR (Sone, 2013).

The mechanism that specifies olfactory sensory neurons to express only one odorant receptor (OR) from a large repertoire is critical for odor discrimination but poorly understood. This study describes the first comprehensive analysis of OR expression regulation in Drosophila. A systematic, RNAi-mediated knock down of most of the predicted transcription factors identified an essential function of acj6, E93, Fer1, onecut, sim, xbp1, and zf30c in the regulation of more than 30 ORs. These regulatory factors are differentially expressed in antennal sensory neuron classes and specifically required for the adult expression of ORs. A systematic analysis reveals not only that combinations of these seven factors are necessary for receptor gene expression but also a prominent role for transcriptional repression in preventing ectopic receptor expression. Such regulation is supported by bioinformatics and OR promoter analyses, which uncovered a common promoter structure with distal repressive and proximal activating regions. Thus, these data provide insight into how combinatorial activation and repression can allow a small number of transcription factors to specify a large repertoire of neuron classes in the olfactory system (Jafari, 2012).

Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events,
through local cell-cell communication. pecanex

(pcx) encodes a multi-pass transmembrane protein of unknown function, widely
found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. This study has established that
pcx is a component of the N-signaling pathway. pcx is required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function results in enlargement of the ER that is not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppresses the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes is induced by overexpression of O-fucosyltransferase, an N-specific chaperone. Taking these results together, it is speculated that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. These results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity (Yamakawa, 2012).

Transcriptional regulation by CHIP/LDB complexes

It is increasingly clear that transcription factors play versatile roles in turning genes 'on' or 'off' depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. This study used the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, 189 down-stream targets of CHIP/LDB were identified; these genes are enriched for the binding sites of Apterous (AP) and Pannier (PNR), two well studied transcription factors associated with CHIP/LDB complexes. Extensive genetic screens were performed and target genes were identified that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing, novel roles were uncovered for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, these results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development (Bronstein, 2011).

Drosophila SSDP was identified on the basis of its ability to bind the nuclear adaptor protein CHIP/LDB. Both nuclear localization of SSDP and its ability to modulate the transcription activity of the CHIP-AP complex during wing development depend on its interaction with CHIP/LDB. This study implemented a combination of molecular, bioinformatic and genetic approaches that allowed has led to insight into the effect of SSDP on the transcriptional activity of CHIP/LDB complexes and their role in development. A genome wide screen was conducted for SSDP target genes in Drosophila using expression microarrays with mRNA isolated from larvae bearing hypomorphic alleles of ssdp. Analysis of transcription factor binding site enrichment served as an orthogonal assay that validates and extends the microarray results and thus contributes to understanding of the relation between the CHIP-AP and CHIP-PNR transcription complexes in specific tissues (e.g., wing and thorax) (Bronstein, 2011).

SSDP proteins directly bind DNA and mouse SSDP1 activates the expression of a reporter gene in both yeast and mammalian cells indicating that it is capable of regulating transcription activity. Enrichment was found for SSDP binding sites upstream of the genes identified in the microarray experiments on flies lacking SSDP. Moreover, in agreement with the positive transcriptional role of SSDP, enrichment for SSDP binding sites was restricted to the genes showing decreased expression in mutants. This strongly suggests that a significant number of these genes are bona fide SSDP target genes (Bronstein, 2011).

Consistent with the involvement of SSDP with the CHIP-AP complex, it was found that upstream regulatory regions of the SSDP putative target genes are also enriched for the AP binding site and the SSDP binding site. These sites are likely to be functionally significant, since loss of ssdp enhances the wing notching phenotype of a dominant allele of ap. Additionally, over-expression of Dlmo, whose product negatively regulates the CHIP-AP complex, also interacts with mutants of SSDP target genes, demonstrating that SSDP target genes are involved in the CHIP-AP pathway. The efficiency of finding genetic interactions among the genes differentially expressed in the microarray experiments, demonstrated the power of this approach. Specifically, 72% of the loci tested with DlmoBx2 is more than an order of magnitude higher than an EP insertion screen (1.3% interacting) in a DlmoBx1 sensitized background. Combined microarray and genetic loss of function screen allowed the identification of a similar number of Dlmo-interacting genes by screening a much smaller group of putative target genes. Of the 35 genes identified by Bejarano (2012), only CG1943 was found in the 189 genes identified in the current microarray screen. This study specifically identified down-stream targets of SSDP, while Bejarano searched for any modifiers of the Dlmo wing notching phenotype and thus uncovered genes that function in other regulatory pathways or genes that are upstream of the CHIP-AP complexes. This may explain the limited overlap between the current results and those of Bejarano (Bronstein, 2011).

In contrast to the enrichment of SSDP binding sites in the genes down-regulated in ssdp mutants, the PNR binding site was enriched specifically in the genes up-regulated in the ssdp mutants. A model is therefore presented in which loss of SSDP disrupts the balance between the CHIP-AP and CHIP-PNR complexes. Mammalian SSDP proteins protect LDB, LHX and LMO proteins from ubiquitination and subsequent proteasome-mediated degradation by interfering with the interaction between LDB and the E3 ubiquitin ligase, RLIM. It is therefore possible that in the absence of SSDP proteins, CHIP/LDB and LMO can escape degradation by interacting with GATA and beta-HLH proteins that are not subjected to proteasome-mediated regulation. The N-terminus of CHIP/LDB proteins is responsible for interaction with both PNR and RLIM. Thus, PNR/GATA proteins may partially interfere with the interaction between CHIP/LDB and RLIM making the CHIP/LDB-PNR/GATA complex more resistant to proteasome regulation and less dependant on the levels of SSDP proteins then the CHIP/LDB-LHX/AP complex (Bronstein, 2011).

According to the current model, in cells where both the CHIP-AP and CHIP-PNR complexes are active, loss of SSDP should result in the same phenotype as over-expression of PNR. Indeed, it was found that ssdpL7/+ flies display duplications of scutellar sensory bristles, similar to gain of function mutations in pnr. In addition, lowered levels of pnr in ssdpL7/+; pnrVX6/+ flies suppresses scutellar bristle duplication. This indicates that the duplicated scutellar bristle phenotype of ssdpL7/+ flies depends on the presence of PNR. As predicted by the model, since both AP and PNR regulate bristle formation, the functional interactions between SSDP target genes and ssdpL7 and/or Chipe5.5 resulted in either suppression or enhancement of the duplicated scutellar bristle phenotype (Bronstein, 2011).

These results in flies indicate that SSDP contributes differentially to CHIP/LDB complexes containing AP versus PNR. By contrast, mouse SSDP proteins positively contribute to the transcription activity and assembly of both LDB-GATA and LDB-LHX complexes, but the relative contribution of mammalian SSDP proteins to LDB complexes containing LHX proteins versus GATA proteins has not been specifically examined. It is possible that SSDP alters the balance of LIM-based CHIP/LDB complexes and GATA-containing CHIP/LDB complexes in the development of mice, as occurs in flies (Bronstein, 2011).

The search for enrichment of transcription factor binding sites upstream of the putative SSDP target genes identified additional transcription factors that may warrant future study. Some of these factors are associated with SSDP and CHIP/LDB complexes. For example, the binding sites for PNR and ZESTE (Z) were both enriched in the up-regulated putative SSDP target genes. This is in agreement with previous studies showing that Z can recruit the BRAHMA (BRM, the Drosophila homolog of the yeast SWI2/SNF2 gene) complex via its member OSA, which together negatively regulate the CHIP-PNR complex during sensory bristle formation through direct and simultaneous binding of OSA to both CHIP and PNR (Bronstein, 2011).

Some of the additional regulatory inputs at SSDP target genes may be evolutionarily conserved. For example, enrichment of STAT92E and SSDP binding sites was found in the down-regulated SSDP target genes. This may be significant, as a known role of ssdp is regulation of the JAK/STAT pathway during Drosophila eye development. Interestingly, mammalian STAT1 confers an anti-proliferative response to IFN-γ signaling by inhibition of c-myc expression. Similarly, expression of mammalian SSDP2 in human acute myelogenous leukemia cells and prostate cancer cells leads to cell cycle arrest and inhibits proliferation accompanied by down-regulation of C-MYC. These findings indicate that both in Drosophila and in mammals SSDP and STAT proteins have similar functions and may share common target genes (Bronstein, 2011).

While the transcription factor binding site analysis utilized all of the 189 putative SSDP target genes, genetic screens were conducted on a subset of them due to the availability of mutants. This suggests that more genetic interactions will be found among the untested genes. Even among this more limited subset, there are interesting new stories that suggest future experimental directions. For example, an insertion mutation in the Xbp1 gene suppressed the duplicated scutellar bristle phenotype characteristic of ssdpL7/+ and Chipe5.5/+ flies, indicating that XBP1 contributes positively to bristle formation. In contrast, when Xbp1 was silenced in ap-expressing cells both the wings and the scutum displayed a marked excess of sensory bristles while the scutellum was not affected. These results suggest that in the wing and scutum XBP1 acts as a negative regulator of bristle formation. Silencing of Xbp1 in pnr-expressing cells caused a similar excess of bristle on the scutum, accompanied by a reduced number of scutellar bristles, further emphasizing the opposing effects of XBP1 in these two distinct parts of the thorax. Such contrasting phenotypes have been previously documented for several pnr mutants as well. In flies and mammals XBP1 regulates the ER stress response, also termed the unfolded protein response (UPR). Since one of the functions of the ER is the production of secreted proteins, UPR-related pathways are widely utilized during the normal differentiation of many specialized secretory cells. In this respect it would be interesting to examine whether SSDP and CHIP/LDB complexes affect the production of secreted morphogens, such as Wingless (WG), the secreted ligands of the EGFR receptor, Spitz (SPI) and Argos (AOS), or the secreted Notch binding protein Scabrous (SCA) via XBP1 during wing and sensory bristle formation. Alternatively, the transcription factor XBP1 may directly regulate the expression of genes required for differentiation of the wing and sensory bristles. Indeed, carbohydrate ingestion induces XBP1 in the liver of mice, which in turn directly regulates the expression of genes involved in fatty acid synthesis. This role of XBP1 is independent of UPR activation and is not due to altered protein secretory function. Curiously, the two GO function categories 'cellular carbohydrate metabolism' and 'cellular lipid metabolism' which are enriched among Xbp1 target genes in mouse skeletal muscle and secretory cells were also enriched in the list of putative SSDP target genes. Whether this reflects a secondary effect due to the down-regulation of Xbp1 in ssdp mutants or a direct regulation of these processes by SSDP is yet to be determined (Bronstein, 2011).

Additional novel functions for CHIP/LDB complexes are implied by the results regarding the Gs-alpha60A (a.k.a. CG2835) gene. G protein coupled receptors are important regulators of development by for example, signaling via the protein kinase A (PKA) pathway. Activation or inhibition of PKA signaling during pupal wing maturation perturb proper adhesion of dorso-ventral wing surfaces resulting in wing blistering. This phenotype may be due to miss-regulation of wing epithelial cell death in ap-expressing cells. Interestingly, similar wing blisters occur in the wing of DlmoBx2 flies. Moreover, it was found that mutant alleles of Gs-alpha60A enhanced the wing blistering phenotype of DlmoBx2. Silencing of G-salpha60A in ap-expressing cells caused a curled wing phenotype. Such a phenotype can result from differences in the size of the dorsal and ventral wing blade surfaces. In addition, silencing of this gene in pnr-expressing cells caused the posterior pair of scutellar bristles to form in reversed orientation. Bristle orientation have been proposed to be regulated by planar cell polarity genes. Taken together these results point to novel aspects of regulation of wing and sensory bristle development by SSDP and CHIP/LDB complexes mediated by G-alpha proteins (Bronstein, 2011).

This genome-wide expression profiling and bioinformatics analysis of ssdp mutant larvae, combined with genetic screens resulted in gained insight into the intricate context-dependent transcriptional regulation by CHIP/LDB complexes. It was possible to identify 28 putative SSDP target genes that are involved in wing development and 23 putative SSDP target genes that play a role in scutellar bristle formation. Examination of two of these, xbp1 and Gs-alpha60A, suggests novel aspects of developmental regulation such as the involvement of SSDP and CHIP/LDB complexes in ER function and PKA signaling. Furthermore, it was shown that SSDP proteins contribute differentially to transcription activity, and probably to the balance in formation of CHIP-AP and CHIP-PNR complexes. Furthermore potential novel partners of SSDP in regulating transcription of downstream genes during fly development were. It stands to reason that an extension of the genetic analysis to mammals and other vertebrates will reveal a host of additional functions of SSDP and CHIP/LDB during the multifaceted process of transcriptional regulation that underlies the development of multicellular organisms (Bronstein, 2011).

Unfolded protein response in a Drosophila model for retinal degeneration

Stress in the endoplasmic reticulum (ER stress) and its cellular response, the unfolded protein response (UPR), are implicated in a wide variety of diseases, but its significance in many disorders remains to be validated in vivo. This study analyzed a branch of the UPR mediated by xbp1 in Drosophila to establish its role in neurodegenerative diseases. The Drosophila xbp1 mRNA undergoes ire-1-mediated unconventional splicing in response to ER stress, and this property was used to develop a specific UPR marker, xbp1-EGFP, in which EGFP is expressed in frame only after ER stress. xbp1-EGFP responds specifically to ER stress, but not to proteins that form cytoplasmic aggregates. The ire-1/xbp1 pathway regulates heat shock cognate protein 3 (hsc3), an ER chaperone. xbp1 splicing and hsc3 induction occur in the retina of ninaEG69D−/+, a Drosophila model for autosomal dominant retinitis pigmentosa (ADRP), and reduction of xbp1 gene dosage accelerates retinal degeneration of these animals. These results demonstrate the role of the UPR in the Drosophila ADRP model and open new opportunities for examining the UPR in other Drosophila disease models (Ryoo, 2007a).

The endoplasmic reticulum (ER) is the cellular organelle where proteins destined for secretion or for diverse subcellular localizations are synthesized and acquire their correct conformation. Perturbations of the environment normally required for protein folding in the ER, or production of large amounts of misfolded proteins exceeding the functional capacity of the organelle, trigger a physiological response in the cell, collectively known as the unfolded protein response (UPR). The UPR serves to cope up with ER stress by transcriptionally regulating ER chaperones and other ER-resident proteins, attenuating the overall translation rate and increasing the degradation of misfolded ER proteins (Ryoo, 2007a).

The mechanisms leading to the UPR activation and its short-term response in regulating gene expression are well characterized in various organisms. In Saccharomyces cerevisiae, unfolded proteins in the ER cause oligomerization of the ER transmembrane protein Ire-1. Upon oligomerization, the endoribonuclease activity of Ire-1 is activated, which catalyzes the unconventional splicing of hac-1 mRNA. Splicing of hac-1 mRNA allows its translation and its protein product acts as a transcription factor, by binding to DNA motifs collectively called UPRE. This leads to the induction of proteins that help to alleviate ER stress. Ire-1 in Caenorhabditis elegans and mammals plays a similar role, by splicing the mRNA of xbp1. Other branches of the UPR include transcription factors ATF4 and ATF6, as well as nontranscriptional mechanisms that reduce the overall amount of misfolded proteins in the ER. This occurs in part through the attenuation of protein translation through PERK, an ER transmembrane kinase, and enhanced rate of protein degradation, also known as ERAD (ER-associated degradation). PERK and the components of the ERAD machinery are activated in response to ER stress (Ryoo, 2007a and references therein).

Whereas transient ER stress can be alleviated by the UPR, a prolonged condition of ER stress can trigger apoptosis. In a mouse model of Pelizaeus-Merzbacher disease (PMD), misfolded proteolipid protein in the ER triggers apoptosis of oligodendrocytes, and several components of the UPR have been shown to play a protective role against the progression of the disease. In contrast, certain components of the UPR, including the PERK/ATF4/CHOP branch, are implicated to play a proapoptotic role. Additionally, numerous cell culture studies implicate the UPR in many disorders caused by misfolded proteins in the cytoplasm, such as Huntington's and Parkinson's diseases. However, the links between the UPR and a wide variety of neurodegenerative disorders remain indirect and controversial, in part, owing to limitations of existing animal model systems (Ryoo, 2007a).

To better understand the role of the UPR in disease, this study focused on a set of alleles in Drosophila ninaE (or rhodopsin-1 (Rh-1)), which have molecular and phenotypic characteristics identical to those found in class III autosomal dominant retinitis pigmentosa (ADRP). These alleles have mutations either in transmembrane domains or in extracellular loops that are predicted to disrupt Rh-1 folding properties. ADRP-afflicted individuals, in both humans and Drosophila, show late-onset retinal degeneration, which occurs through the activation of apoptosis. Inhibiting caspases blocks retinal degeneration and blindness in the Drosophila model, demonstrating that apoptosis is the main cause of the disease (Ryoo, 2007a).

This study examined whether the UPR contributes to the progression of retinal degeneration in class III ADRP model of Drosophila. xbp1 splicing and the induction of an ER chaperone, heat shock cognate protein 3 (hsc3), are shown to occur in response to ER stress. Using this knowledge, an xbp1-EGFP fusion transgene was devised as an in vivo marker of ER stress, designed to have EGFP expressed in frame with xbp1 only after Ire-1-mediated splicing occurs. xbp1-EGFP splicing occurs after mutant Rh-1 expression, but is not detectable in response to polyglutamine repeats or tau R406W, which cause neurodegenerative disorders in humans by forming cytoplasmic protein aggregates. xbp1-EGFP splicing also occurs in the Drosophila model of ADRP, and xbp1 has a protective role against retinal degeneration. These results demonstrate that ER stress occurs in the Drosophila ADRP model and suggest possible mechanisms by which apoptosis is activated in response to mutant Rh-1 molecules (Ryoo, 2007a).

ER stress has been implicated in a wide variety of human diseases, including many neurodegenerative disorders and cancer, but relatively few have been validated in animal models. This study has shown that the basic UPR pathway is conserved from yeast, C. elegans, Drosophila and mammals. Moreover, it was demonstrated that the UPR is activated in the ADRP model of Drosophila, and this plays a protective role against the progression of retinal degeneration (Ryoo, 2007a).

xbp1 splicing is a specific indicator of the UPR signaling in Drosophila. Using the xbp1-EGFP fusion construct, this study detected xbp1 splicing in response to ER-stress-causing agents (DTT, thapsigargin and tunicamycin), ectopic expression mutant Rh-1, and in the Drosophila model of ADRP. However, in vivo experiments did not support previous observations that cytoplasmic aggregates can also cause ER stress indirectly, through compromising the proteasome capacity. xbp1 activation in the ADRP model can account for many phenotypes previously reported. One such phenotype is the dramatic enlargement of the ER network. As studies in other organisms have shown that the ire-1/xbp1 branch of UPR promotes ER membrane biogenesis, it is most likely that the enlarged ER-network biogenesis of ninaEG69D−/+ retina is due to Drosophila xbp1 activation. In contrast to the stressed cells, significant levels of xbp1 (or xbp1-EGFP) splicing were not detected in unstressed developing tissues of embryos or late third instar larvae. This is consistent with previous studies conducted in transgenic mice harboring an xbp1-GFP(venus) construct similar to the one used in this study. In mice, xbp1 splicing was not detected in embryonic or postnatal stages, but only after late postnatal stage (16 days or older) and only in a few tissues. A small number of cells showing xbp1-EGFP splicing was observed in earlier stage larvae, indicating that occasional xbp1 splicing occurs during normal development. As xbp1 mutation is recessive lethal, the low level of natural xbp1 splicing may account for the requirement of this gene in Drosophila development. It cannot be excluded, however, that the unspliced RA form of xbp1 also plays a role during development, accounting for the lethality of xbp1-deficient animals. xbp1 is required during embryonic development in mammals, but not in C. elegans, where the animals can complete their developmental program without xbp1. In addition to xbp1, hsc3 and ire-1, other genes homologous to those implicated in the UPR are found in the Drosophila genome. These include the ER transmembrane kinase perk and the ER tethered transcription factor ATF6 (annotated as CG3136). Although their in vivo function has not been analyzed in Drosophila, their presence and high sequence homology suggest a conserved function in the UPR (Ryoo, 2007a).

Previous studies demonstrated that the class III ninaE alleles show caspase-dependent cell death. The underlying mechanism by which caspases become active in these cells remains unclear, but the finding of the UPR activation in these cells provides at least three models. First model is based on the observation that JNK signaling is activated as part of the UPR. In this model, activation of the UPR stimulates Ire-1/TRAF interaction, independent of xbp1 mRNA splicing, leading to JNK activation and apoptosis. In fact, Ire-1/TRAF/JNK signaling is required for apoptosis in response to poly-glutamine repeat accumulation in cultured cells. In addition, enhanced JNK signaling is detected in the retinas of a class III ninaE allele in Drosophila. Finally, activation of JNK signaling in Drosophila results in the induction of the proapoptotic gene hid. The second model is based on the observations that Ca2+ release from the ER can activate caspase activation and cell death. It is possible that the disruption of ER homeostasis by the accumulation of misfolded proteins in the ER results in the release of Ca2+ into the cytoplasm, activating a proteolytic cascade leading to caspase activation. Third, as CHOP has been implicated to mediate ER-stress-triggered apoptosis in mammals, a similar mechanism may exist in Drosophila. However, there are no obvious homologs of CHOP in the Drosophila genome. Whether these models account for the death of class III ninaE mutant photoreceptors awaits further studies (Ryoo, 2007a).

Drosophila provides a unique advantage as a model for studying human diseases associated with ER-stress-triggered cell death, as the mechanisms of stress-provoked caspase activation are largely conserved between the two species. In addition, the short lifespan of Drosophila, combined with a similarly accelerated rate of chronic disease manifestation, may help validate the in vivo significance of the UPR in a growing list of disorders, ranging from neurodegenerative disease involving cytoplasmic protein aggregates, hypoxia during cancer progression and p53-induced cell death. In this regard, the present work on the role of the UPR in Drosophila retinal degeneration may provide a framework for further investigations into the UPR in human disease (Ryoo, 2007a).

The ability of cells to survive and recover from deteriorating effects of endoplasmic reticulum (ER) stress relies on the unfolded protein response (UPR). The signaling pathway of Ire1p mediate mRNA splicing plays a divergant role in UPR responses in different organisms from yeast to mammals. This report that Ire1p mediated XBP1 mRNA splicing mechanism is extremely conserved and exerts a critical role for modulating Xbp1 protein synthesis in Drosophila melanogaster. This system is operative in Drosophila S2 cells as a prominent mechanism to mediate transcriptional activation of UPR responsive genes during ER stress (Plongthongkum, 2007).

Transcriptional activation of genes encoding molecular
chaperones and folding enzymes during ER stress is a
major response in eukaryotic cells to mount the stress for
their survival. This study characterized the UPR
pathway in Drosophila S2 cells. Drosophila Xbp1 is a bzip
transcription factor that showed a very low homology in
lysine, leucine rich region to its counterparts in mammal
or worm. Interestingly, its coding sequence is organized in a highly conserved fashion.
The fly XBP1 mRNA
contains two overlapping ORFs. The ORF2 is in +1 frame
relative to the ORF1. These ORFs were joined into the
same reading frame during ER stress prior translation by
eliminating the 23 nucleotide intron through an unusual
splicing mechanism. The nucleotide sequence surrounding
this intron showed extremely conserved feature of two 7
nucleotides ring stem loop structures critical for Ire1 mediated
splicing in mammalian cells. This reveals that the
stress regulated mRNA splicing pathway by Ire1p is
remarkably conserved through evolution (Plongthongkum, 2007).

The finding that the endogenous XBP1u mRNA in S2
cells was not translated into protein but it was efficiently
translated in transiently transfected cells providing an
intriguing observation. The ORF2 located further down
stream of the 5' end of the mRNA was simultaneously
translated into protein implicating that the presence of
the intron may form secondary structure which facilitates
internal ribosome entry resembling Internal
Ribosome Entry Site (IRES) of several RNA viruses. Currently there is no explanation why this structure
is not functional in the Drosophila cells. It is possible that
S2 cells produce specific factor(s) which bind and suppress
the translation of the XBP1u mRNA. Albeit translated,
the protein from ORF1 or ORF2 alone exhibits no
activity. It is most likely that the two regions serve different
functions such as DNA binding domain and transcription
activation domain that must be linked to
function (Plongthongkum, 2007).

While the mammalian UPR is a complex coordinating multiple mechanisms of Ire1/XBP1, ATF6, and PERK,
the UPR in Drosophila S2 cells appears simpler, by relying
mainly on Ire1/XBP1 pathway. Suppression of either IRE1
or XBP1 by RNAi completely abolished the UPR response.
Nonetheless, this pathway might not be a simple linear signaling
cascade. Besides inducing genes enriched ER-related
processes during the stress, recent study has demonstrated
additional functions of the insect UPR in mediating rapid
repression of ER-targeted mRNAs. Such functions
requires only Ire1 activity but are independent of Xbp1. Hence
this system would be an interesting model for further elucidating
the mechanism detail and physiological significant
of metazoan Ire1/XBP1 cascade (Plongthongkum, 2007).

The xpb-1 gene is essential for development in Drosophila

This paper reports the characterization of Dxbp-1, the Drosophila homologue of the xpb-1 gene that encodes a "bZIP"-containing transcription factor that plays a key role in the unfolded protein response (UPR), an evolutionarily conserved signalling pathway activated by an overload of misfolded proteins in the endoplasmic reticulum (ER). Dxbp-1 is ubiquitously transcribed, and high levels are found in embryonic salivary glands and in the ovarian follicle cells committed to the synthesis of the respiratory appendages. Loss of function of Dxbp-1 induced a recessive larval lethality, thus, revealing an essential requirement for this gene. The Dxbp-1 transcript was submitted to an "unconventional" splicing that generated a processed Dxbp-1s transcript encoding a DXbp-1 protein isoform, as is the case for yeast, Caenorhabditis elegans and vertebrate hac1/xbp-1 transcripts after UPR activation. However, in the absence of exogenously induced ER stress, the Dxbp-1s transcript was also detectable not only throughout embryonic and larval development but also in adults with a high level of accumulation in the male sexual apparatus and, to a lesser extent, in the salivary glands of the third-instar larvae. Using a Dxbp-1:GFP transgene as an in vivo reporter for Dxbp-1 mRNA unconventional splicing, it was confirmed that Dxbp-1 processing took place in the salivary glands of the third-instar larvae. The Dxbp-1 gene appears, thus, to play an essential role during the development of Drosophila, hypothetically by stimulating the folding capacities of the ER in cells committed to intense secretory activities (Souid, 2007).